Bone Marrow-derived Monocytes Research Articles

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival.

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.

Evidence of CCR2-independent transmigration of Ly6Chi monocytes into the brain after permanent cerebral ischemia in mice

Previously we showed that INCB3344, a CCR2 antagonist, inhibits transmigration of Ly6Chi monocytes into the brain after ischemia-reperfusion. Here we tested the effect of CCR2 inhibition during permanent cerebral ischemia. Mice were administered either vehicle (dimethyl sulfoxide/carboxymethylcellulose) or INCB3344 (30 or 100mg/kg IP) 1h before middle cerebral artery occlusion and at 2 and 6h after the initiation of ischemia. After 24h, we assessed functional outcome, infarct volume and quantified immune cells in blood and brain. The increase in circulating bone marrow-derived Ly6Chi monocytes, but not the infiltration of those cells into the brain, was blocked by the CCR2 antagonist. INCB3344 had no effect on either neurological deficit or infarct volume. Our data confirm that cerebral ischemia triggers a CCR2-dependent increase in circulating Ly6Chi monocytes, but suggest that in the absence of reperfusion these cells may transmigrate into the ischemic brain in a CCR2-independent manner.

Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells.

Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them.

Induction of protumoral CD11c(high) macrophages by glioma cancer stem cells through GM-CSF.

Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor-associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow-derived monocytes. CSC-induced macrophages can be separated into two distinct subsets of cells, CD11c(low) and CD11c(high) cells. Interestingly, only the CD11c(high) subset of cells have protumoral activity, as shown by intracranial transplantation into immune-deficient mice together with CSCs. These CD11c(high) macrophages were observed in the tumor formed by co-transplantation with CSCs. Furthermore, CSCs produced GM-CSF and anti-GM-CSF antibody inhibited CSC-induced TAM development. In conclusion, CSCs have the ability to self-create their own niche involving TAMs through CSC-derived GM-CSF, which can thus be a therapeutic target in view of CSC niche disruption.

Abstract B180: Cytokine regulation of PD-L1 expression on tumor-associated myeloid cells

Abstract Background and Significance: PD-L1 is an immune checkpoint protein that has emerged as a major signaling molecule involved with tumor escape from T cell immune responses. Studies have shown that intra-tumoral expression of PD-L1 can inhibit antitumor immune responses. However, it has recently been shown in a study using human patient tumor samples that expression of PD-L1 on myeloid cells from the tumor is a stronger indicator of prognosis than tumor cell PD-L1 expression. Therefore, it is important to understand the factors that govern the regulation of PD-L1 expression on tumor-infiltrating myeloid cells. We initially investigated the role of IFN-γ in regulating macrophage PD-L1 expression using the murine B16 model and found that IFN-γ did not affect PD-L1 expression by tumor macrophages in this model. Thus, we conducted studies to identify other cytokines that may play an important role in regulating PD-L1 expression on myeloid cells in the tumor microenvironment. Methods: In our preliminary studies, IFN-γ knockout and wild-type B57BL/6 mice were inoculated with B16 melanoma cells and tumor tissue was harvested and stained for flow cytometric analysis of PD-L1 expression on tumor and myeloid cells. In vitro cell staining experiments were conducted using immature bone marrow monocytes (CD11b+/ Ly6C+/Ly6G-), circulating blood monocytes (CD11b+/F4-80+/ Ly6C+/Ly6G-), and macrophages in tumor tissue (CD11b+/F4-80+) that were stained for baseline expression levels of PD-L1. Bone marrow-derived monocytes were then co-cultured with B16 tumor cells, B16 supernatants, or stimulated with recombinant cytokines for flow cytometric analysis to detect changes in PD-L1 expression. The supernatants from co-cultured cells were also collected for identification and quantification of cytokines by ELISA. Results: We found that immature bone marrow monocytes in tumor-bearing mice had low levels of PD-L1 expression, while higher levels of expression were observed on monocytes in circulation. In contrast, macrophages found in tumor tissues expressed much higher levels of PD-L1 than circulating monocytes, implying upregulation by the tumor microenvironment. Using in vitro co-culture systems, we demonstrated co-culture with tumor cells strongly induced PD-L1 upregulation by bone marrow-derived monocytes, and that this effect was mediated by a soluble factor. Studies are currently in progress to identify the cytokines secreted by tumor cells that are responsible for PD-L1 upregulation. Conclusion: Cytokines other than IFN-γ are responsible for upregulation of PD-L1 expression by tumor-associated macrophages, but in vitro and in vivo. Thus, PD-L1 expression by tumor macrophages appears to be regulated in a different manner than by tumor cells themselves. Citation Format: Genevieve Hartley, Dan Regan, Amanda Guth, Steven Dow. Cytokine regulation of PD-L1 expression on tumor-associated myeloid cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B180.

3D Printing of Bone-Templated Scaffolds

SJ Fernando1, JA Sterling2,3, SA Guelcher1 1Vanderbilt University Medical Center, Department of Chemical and Biomolecular Engineering, 2 Veterans' Affairs Tennessee Valley Healthcare System, Nashville, 3Vanderbilt University, Department of Clinical Pharmacology Statement of Purpose: Biomimetic 3D tissue engineered systems have been proposed for investigating molecular mechanisms of disease progression and for screening drugs [1]. We have utilized 3D printing technology to investigate how the mechanical and topological properties of the bone microenvironment influence both cellular interactions and tumor progression in bone. Trabecular curvature, pore size, and pore shape have also been reported to affect the rate of new bone formation. Cells sense and respond to radii of curvature larger than themselves, and the rate of new bone formation increases with the curvature of the surface [2]. The Structure Model Index (SMI) parameter characterizes the structure of trabecular bone, with 0 representing plate-like (e.g., 2D) structures and 3 representing rod-like structures.[3] The trabecular structure largely varies between values of 0 and 3, rather than being predominantly plates or rods. Vertebral trabeculae have an SMI typically closer to a rod-like structure (SMI=3), whereas femoral trabeculae have structure closer to plates (SMI=0). We reason that 3D scaffolds replicating the mechanical and topological properties of bone at different anatomic sites will enable the development of 3D cell culture models for investigating the spatio-temporal dynamics of both bone cellular physiology and cancer progression [1]. We have developed a fabrication process in which wax templates of trabecular bone are prepared by a 3D inkjet printer and subsequently filled with reactive polyurethanes to create scaffolds with elastic modulus and SMI comparable to human bone. Methods: Human cadaver samples from the proximal tibia, proximal femur, and lumbar vertebrae were obtained from the Program in Advanced Anatomy and Simulated Skills Program at Vanderbilt. Isolated tissue samples were imaged using uCT technology from Scanco Inc. Using Scanco software the scans were converted into STL images, a format representative of the surface of bone uCT images, which specifically captures the trabecular architecture. These images were inverted using the same software to create a representation of the trabecular spacing (Tb.Sp.) The STL images of Tb.SP were then uploaded into the Solidscape Studio 3Z Printer for fabrication. The resulting wax molds were filled with a reactive two-component polyurethane (PUR) composed of lysine diisocyanate (LDI), a poly(e-caprolactone-coglycolide) triol (Mw=300 or 3000 g/mol), and iron(III) acetylacetonate (FeAA) catalyst. The polyol (900 Da) was prepared from glycerol starter and backbone comprising 70wt% ɛ-caprolactone, 20wt% glycolide, and 10wt% D,L-lactide. The mixture was cured overnight under vacuum at 80° C. The cured polyurethane structure was extracted from the wax mold using acetone solution to dissolve the wax (sulphonamide derivatives, polyester resin, and benzoate derivatives,) and dried under vacuum overnight. The resulting structures were imaged by SEM and uCT. Nanoindentation was performed on 2D films and on the 3D biomimetic PUR scaffolds. Rat bone marrow-derived stromal cells and monocytes, as well as bone-metastatic MDA-MB-231 breast cancer cells were incubated with the scaffolds under standard cell culture conditions.

Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay.

The NLRP3 inflammasome is assembled in macrophages and monocytes in response to inflammatory and danger stimuli. The atypical nature of the NLRP3 complex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein-protein interactions in tissue and cell samples. Two primary antibodies raised in different species detect the two proteins of interest. When the proteins are in close proximity, secondary antibodies conjugated with specific DNA probes hybridize with linking oligonucleotides to form a DNA bridge between the two proteins. Amplification of the DNA bridge then facilitates detection by microscopy using fluorescence probes. Here, we describe application of in situ PLA to detect NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages and human monocyte cell line THP1.

Diabetes-Induced Oxidative Stress in Endothelial Progenitor Cells May Be Sustained by a Positive Feedback Loop Involving High Mobility Group Box-1.

Oxidative stress is considered to be a critical factor in diabetes-induced endothelial progenitor cell (EPC) dysfunction, although the underlying mechanisms are not fully understood. In this study, we investigated the role of high mobility group box-1 (HMGB-1) in diabetes-induced oxidative stress. HMGB-1 was upregulated in both serum and bone marrow-derived monocytes from diabetic mice compared with control mice. In vitro, advanced glycation end productions (AGEs) induced, expression of HMGB-1 in EPCs and in cell culture supernatants in a dose-dependent manner. However, inhibition of oxidative stress with N-acetylcysteine (NAC) partially inhibited the induction of HMGB-1 induced by AGEs. Furthermore, p66shc expression in EPCs induced by AGEs was abrogated by incubation with glycyrrhizin (Gly), while increased superoxide dismutase (SOD) activity in cell culture supernatants was observed in the Gly treated group. Thus, HMGB-1 may play an important role in diabetes-induced oxidative stress in EPCs via a positive feedback loop involving the AGE/reactive oxygen species/HMGB-1 pathway.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

Abstract B64: CSF1R-dependent tumor-associated macrophages arise from bone marrow-derived monocytes and promote gastrointestinal stromal tumor development

Abstract Introduction: Gastrointestinal stromal tumors (GISTs) are densely infiltrated with tumor-associated macrophages (TAMs), which we have previously shown to exhibit an M1, inflammatory phenotype in established, untreated tumors in mice and humans. Since TAMs in most tumors are reported to be M2, we sought to determine the role of TAMs in early GIST development and the mechanism of TAM recruitment. Methods: Genetically engineered KitV558del/+ mice that develop GIST were treated as adults and from birth with PLX5622, a small molecule inhibitor of colony stimulating factor 1 receptor (CSF1R). Tumors were weighed and analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. Adoptive transfer experiments utilized tail vein injection of freshly harvested CD45.1+, CFSE-labeled bone marrow cells 2 days following withdrawal of CSF1R inhibition. Cell proliferation was determined by Ki67 staining. Results: Tumors from KitV558del/+ mice treated with PLX5622 from birth for 4 weeks showed an 85% reduction of TAMs and a 40% decrease in size (n=8/group, p<0.05). TAMs from untreated 2 week old developing GIST mouse tumors tended to be more M2-like with 2.5x lower CD11c and 1.25x lower MHCII, while the M2 markers CD206 and MSR1 were increased 1.5x and 3x, respectively. Likewise, 3 days following withdrawal of PLX5622, newly recruited TAMs displayed a 20% reduction of MHCII+ cells (p<0.05). Furthermore, following PLX5622 withdrawal, flow cytometry revealed that TAM repopulation was preceded by an initial surge in Ly6Chi monocytes in the blood and the tumor. Subsequently, there was a reciprocal decrease of blood monocytes as the F4/80hi TAM population expanded to baseline levels of 35-40% of leukocytes over the course of 7 days. TAMs exhibited increased proliferation (55% Ki67+) compared to monocytes (17% Ki67+) and Kit+ tumor cells (2.5% Ki67+) 4 days after withdrawal of CSF1R inhibition. Following adoptive transfer of CD45.1+, CFSE-labeled bone marrow, the labeled cells showed an increasing TAM:monocyte ratio over time (2 on day 3, 7 on day 5, and 68 on day 10). Conclusion: Although TAMs infiltrating established GISTs in adult mice exhibit an M1-like, anti-tumor phenotype, newly recruited TAMs in tumor development or TAMs after CSF1R inhibition are relatively M2-like. Within the tumor, bone marrow-derived monocytes differentiate into CSF1R-dependent TAMs, which proliferate to fully repopulate the tumor. Citation Format: Michael J. Beckman, Michael Cavnar, Adrian Seifert, Juan Santamaria-Barria, Jennifer Zhang, Adam Levy, Ferdinand Rossi, Shan Zeng, Ronald P. DeMatteo. CSF1R-dependent tumor-associated macrophages arise from bone marrow-derived monocytes and promote gastrointestinal stromal tumor development. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B64.

Autophagy-induced RelB/p52 activation mediates tumour-associated macrophage repolarisation and suppression of hepatocellular carcinoma by natural compound baicalin

The plasticity of tumour-associated macrophages (TAMs) has implicated an influential role in hepatocellular carcinoma (HCC). Repolarisation of TAM towards M1 phenotype characterises an immune-competent microenvironment that favours tumour regression. To investigate the role and mechanism of TAM repolarisation in suppression of HCC by a natural compound baicalin, Orthotopic HCC implantation model was used to investigate the effect of baicalin on HCC; liposome-clodronate was introduced to suppress macrophage populations in mice; bone marrow-derived monocytes (BMDMs) were induced to unpolarised, M1-like, M2-like macrophages and TAM using different conditioned medium. We observed that oral administration of baicalin (50 mg/kg) completely blocked orthotopic growth of implanted HCC. Suppression of HCC by baicalin was diminished when mice macrophage was removed by clodronate treatment. Baicalin induced repolarisation of TAM to M1-like phenotype without specific toxicity to either phenotype of macrophages. Baicalin initiated TAM reprogramming to M1-like macrophage, and promoted pro-inflammatory cytokines production. Co-culturing of HCC cells with baicalin-treated TAMs resulted in reduced proliferation and motility in HCC. Baicalin had minimal effect on derivation of macrophage polarisation factors by HCC cells, while directly induced repolarisation of TAM and M2-like macrophage. This effect was associated with elevated autophagy, and transcriptional activation of RelB/p52 pathway. Suppression of autophagy or RelB abolished skewing of baicalin-treated TAM. Autophagic degradation of TRAF2 in baicalin-treated TAM might be responsible for RelB/p52 activation. Our findings unveil the essential role of TAM repolarisation in suppressive effect of baicalin on HCC, which requires autophagy-associated activation of RelB/p52.

Irradiation Enhances the Ability of Monocytes as Nanoparticle Carrier for Cancer Therapy

The tumor-homing ability of monocytes renders them a potential cellular delivery system for alternative cancer therapies, although their migratory ability can be impaired following reagent uptake. Approaches that enhance monocyte tumor homing and promote their migration will improve the clinical value of these cells as cellular carriers. Previous studies have shown that irradiation (IR) can promote macrophage aggregation in hypoxic regions. To investigate whether IR enhances the infiltration of bone marrow-derived monocytes (BMDMs) into tumors, the infiltration of BMDMs from GFP-transgenic mice in a murine prostate adenocarcinoma TRAMP-C1 model was examined by fluorescence microscopy. IR did not increase the number of BMDMs that infiltrated initially, but did increase monocyte retention within IR-treated tumors for up to 2 weeks. We also showed that BMDMs can take up various imaging and therapeutic agents, although the mobility of BMDMs decreased with increasing load. When BMDMs were differentiated in IR-treated tumor-conditioned medium (IR-CM) in vitro, the nanoparticle load-mediated inhibition of migration was attenuated. These IR-CM-differentiated BMDMs delivered polymer vesicles encapsulating doxorubicin to radiation therapy (RT)-induced hypoxic tumor regions, and enhanced the efficacy of RT. The prolonged retention of monocytes within irradiated tumor tissues and the ability of IR-CM to enhance the migratory ability of cargo-laden BMDMs suggest that monocytes pre-conditioned by IR-CM can potentially act as cellular carriers for targeted therapy following conventional RT.

Lanthanum Chloride Attenuates Osteoclast Formation and Function Via the Downregulation of Rankl-Induced Nf-κb and Nfatc1 Activities.

The biological activities of lanthanum chloride (LaCl3 ) and the molecular mechanisms of action underlying its anti-inflammatory, anti-hyperphosphatemic, and osteoblast-enhancing effects have been studied previously, but less is known about the effects of LaCl3 on osteoclasts. The present study used in vivo and in vitro approaches to explore the effects of LaCl3 on osteoclasts and osteolysis. The results indicated that LaCl3 concentrations that were non-cytotoxic to mouse bone marrow-derived monocytes attenuated receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis, bone resorption, mRNA expression of osteoclastogenic genes in these cells, including cathepsin K, calcitonin receptor, and tartrate-resistant acid phosphatase (TRAP). Further, LaCl3 inhibited RANKL-mediated activation of the nuclear factor-κB (NF-κB) signaling pathway, and downregulated mRNA and protein levels of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), and c-fos. In vivo, LaCl3 attenuated titanium (Ti) particle-induced bone loss in a murine calvarial osteolysis model. Histological analyses revealed that LaCl3 ameliorated bone destruction and decreased the number of TRAP-positive osteoclasts in this model. These results demonstrated that LaCl3 inhibited osteoclast formation, function, and osteoclast-specific gene expression in vitro, and attenuated Ti particle-induced mouse calvarial osteolysis in vivo, where the inhibition of NF-κB signaling and downregulation of NFATc1 and c-fos played an important role.

Isolation of mouse bone marrow-derived monocytes

Isolation of mouse bone marrow-derived monocytes

Monocytic delivery of therapeutic oxygen bubbles for dual-modality treatment of tumor hypoxia

Photodynamic therapy (PDT) is a powerful technique photochemically tailored for activating apoptosis of malignant cells. Although PDT has shown promise in several clinical applications, malignant cells in hypoxic regions are often resistant to PDT due to the transport limitation of therapeutics and the oxygen-dependent nature of PDT. Herein, we present an innovative strategy for overcoming the limits of PDT in tumor hypoxia using bone marrow-derived monocytes as cellular vehicles for co-transport of oxygen and red light activatable photosensitizer, chlorin e6 (Ce6). Superparamagnetic iron oxide nanoparticle/Ce6/oxygen-loaded polymer bubbles were prepared and internalized into tumortropic monocytes. These functional bubbles were found harmless to cellular hosts without external triggers. Nevertheless, the therapeutic monocytes exhibited a superior performance in inhibiting tumor growth on Tramp-C1 tumor-bearing mice (C57BL/6J) upon the treatments of tumors with high frequency magnetic field and red light laser (660nm). Histological examinations of the tumor sections confirmed the successful cellular transport of therapeutic payloads to tumor hypoxia and the pronounced antitumor effect elicited by combined hyperthermia/photodynamic therapy along with the additional oxygen supply. This work demonstrates that this oxygen/therapeutic co-delivery via tumortropic monocytes toward tumor hypoxia is promising for improving PDT efficacy.

ID: 25: Substance P as a novel cytokine for induction of alternatively activated M2 polarization and skewing M2 from M1 type of macrophages

We recently showed that intravenous treatment of Substance P (SP) after spinal cord injury increased the number of alternatively activated M2 macrophages at injured lesion of spinal cord and eventually promoted tissue repair. However, it was unclear enhanced M2 polarization of differentiated macrophages is resulted from SP-mediated signaling pathway or accompanied by immune modulatory actions of SP. In the present study, we aim to elucidate the direct effect of SP in M2 polarization in tissue macrophages. Rat macrophages (MO), differentiated with GM-CSF treatment for 5 days on bone marrow-derived monocytes (MO) were prepared. Immunofluorescence staining with specific marker for monocytes and macrophages, MO was characterized as ED-1 (CD68, a marker for macrophages) and CCR7-positive M1-like precursor differentiated from CD14 and CD11b-positive bone marrow monocytes. Administration of SP in MO enhanced the expression of M2 marker, CD206 and CD163 concurrently with the reduction of M1 marker, CCR7 and CD68. M2 polarization of MO in response to SP was completely blocked by NK1-R antagonist, RP67580, and PI3K inhibitor, LY294002. Furthermore, SP also showed transforming ability of M1-polarized MO induced by IFN toward M2 type with induction of CD163 expression. In summary, the results of the present study demonstrate that SP directly polarizes M2 macrophages of MO and IFN-primed M1 type and acts as a Th2 cytokine.

Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity.

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.

FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

BackgroundPeriodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.MethodsMurine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR).ResultsFTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts.ConclusionFTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

Macrophage and epithelial cell H-ferritin expression regulates renal inflammation

Inflammation culminating in fibrosis contributes to progressive kidney disease. Crosstalk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice; a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Utilizing transgenic mice with conditional deletion of FtH in the proximal tubules (FtHPT−/−) or myeloid cells (FtHLysM−/−), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared to wild-type (FtH+/+) controls. However, tubular FtH deletion led to a marked increase in pro-inflammatory macrophages. Furthermore, injured kidneys from FtHPT−/− mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared to kidneys from FtH+/+ and FtHLysM−/− mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscores the critical role of FtH in tubular-macrophage crosstalk during kidney injury.